The objective of the experiments described in this proposal is to determine aspects of the molecular structure of (Na plus K ions) adenosine triphosphatase which permit this enzyme to catalyze the active transport of sodium and potassium across the plasma membrane. The larger subunit of this enzyme, which spans the membrane and contains the active site and the cardiac glycoside receptor, will be cleaved into cyanogen bromide fragments and the order in which these fragments occur in the sequence of the protein will be discovered. It will then be possible to label specifically those regions of this protein which are located within the active site, within the cardiac glycoside site, on the outside of the cell membrane, and on the inside of the cell membrane. The position of each of these regions in the linear sequence of the protein will be assigned by isolating the cyanogen bromide fragments which are labelled in each instance. These results will indicate how this polypeptide is coiled so that it can span the plasma membrane, catalyze active transport of sodium and potassium, and transmit information between the cardiac glycoside receptor on the outside surface of the plasma membrane and the active site at the inside surface of the plasma membrane. In order to discover how many subunits form the active complex and to measure distances between specific points in the protein, chemical crosslinking and fluorescent energy transfer experiments will be attempted. These experiments will increase our understanding of (Na plus K ions) adenosine triphosphatase whose function is of central importance to those physiological mechanisms which pace heartbeat, create the action potentials of the nervous system, and control water and electrolyte fluxes in the kidney and other transporting epithelia. BIBLIOGRAPHIC REFERENCES: Kyte, J., (1976) Immunoferritin determination of the distribution of Na ion plus K ion ATPase over the plasma membranes of renal convoluted tubules I. Distal segment. J. Cell Biol. in the press. Kyte, J., (1976) Immunoferritin determination of the distribution of Na ion plus K ion ATPase over the plasma membranes of renal convoluted tubules II. Proximal segment. J. Cell Biol. in the press.